Amylase assays measure total activity without differentiating the relative contributions of pancreatic- and salivary-type amylase isozymes. Since polyacrylamide electrophoresis allows identification of salivary-and pancreatic-type isoxymes and their respective variants, serum and urine specimens from patients with the clinical diagnoses of mumps (4), pancreatitis (16), or undiagnosed hyperamylasemias (5) were compared with specimens from control subjects. Patients with mumps had elevations of salivary-type isozymes, while those with pancreatitis had elevations of pancreatic-type isozymes. Elevation of salivary-type isozymes was identified in the five patients who had undiagnosed hyperamylasemias; among these, the isozymes of two originated in neoplastic ovarian tissue and those of three, probably in the salivary glands. Amylase isozyme differentiation cannot unamibiguously identify the tissue source of hyperamylasemia. However, in patients whose hyperamylasemia is of unknown etiology or who respond atypically to therapy, amylase electrophoresis provides identification of the elevated isozyme type, thus providing the basis for the rational selection of further diagnostic procedures.